Summary Activation of naive CD8 T cells initiates their differentiation into effector and memory cytotoxic T lymphocytes (CTL) that control infections and tumors using the cytotoxic proteins perforin (Prf1), granzyme B (Gzmb), and effector cytokines such as Ifng. We showed that the transcription factor Runx3 is essential for the transcriptional activation of the Prf1, Gzmb, and Ifng genes upon CD8 T cell activation. In addition, our results showed that Runx3 induces and then cooperates with the T-box transcription factor Eomesodermin. However, the basic principles that guide programming of chromatin structure by these factors to establish and maintain CTL differentiation are unknown. Nucleosomes are the fundamental repeating subunit of chromatin, and they directly bind DNA, which obscures encoded transcription factor binding motifs from their cognate factors. There is very little evidence in mammalian systems to explain how transcription factors invade nucleosomal DNA to bind their cognate sites in vivo in order to change transcriptional programs; both enzymatic nucleosome remodeling and the thermodynamics of nucleosome:DNA and transcription factor:DNA interactions are likely to determine transcription factor binding. To study this fundamental problem, we developed an innovative method to map the in vivo positions and occupancy of nucleosomes on DNA at very high resolution and applied it in the context of a highly tractable cell-culture system that recapitulates important aspects of effector and memory CTL differentiation. Our preliminary studies indicate that nucleosomes undergo striking changes in occupancy during differentiation, and these changes are distinct in effector and memory CTL conditions. Notably, most nucleosomes that are remodeled localize in DNase I hypersensitive (DHS) sites that are physically occupied by Runx3 transcription factors, as judged by chromatin immunoprecipitation. This suggests that Runx3 binding might regulate nucleosome occupancy directly. In this proposal we will test the hypothesis that Runx3 binding controls nucleosome positions and occupancy to establish the underlying chromatin structure that enables CTL differentiation. Our goals are to map Runx3 binding sites genome-wide and to titrate Runx3 expression during CTL differentiation to test whether Runx3 competes with nucleosomes for DNA occupancy in vivo (Aim 1). Next we will determine the genome-wide distribution of chromatin remodeling complexes that use the Brg1 ATPase during CTL differentiation, and clarify whether Runx3 requires its activity to alter nucleosome positions in cis-domains that they co-occupy (Aim 2). Lastly, we will generate and analyze mice lacking one of three important DHS sites in the Prf1 locus that binds Runx3 and that undergoes nucleosome depletion upon CTL differentiation, and determine how Runx3 deficiency affects perforin expression and CTL differentiation during viral infection (Aim 3). Successful completion of these Aims will provide the first look at the affinity chromatin landscape that determines CTL differentiation, and will help to clarify the basic problem relating to how a developmentally regulated transcription factor gains access to its binding sites in chromatin.